Lipocalin 15 in the olfactory mucus is a biomarker for Bowman’s gland activity

Olfactory mucus contributes to the specific functions of the olfactory mucosa, but the composition and source of mucus proteins have not been fully elucidated. In this study, we used comprehensive proteome analysis and identified lipocalin 15 (LCN15), a human-specific lipocalin family protein, as an abundant component of the olfactory mucus. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using a newly generated anti-LCN15 antibody showed that LCN15 was concentrated in olfactory mucus samples, but not in respiratory mucus samples. Immunohistochemical staining using anti-LCN15 antibody revealed that LCN15 localized to the cytokeratin 18-positive Bowman's glands of the olfactory cleft mucosa. Quantitative image analysis revealed that the area of LCN15 immunoreactivity along the olfactory cleft mucosa significantly correlated with the area of neuron-specific Protein-Gene Product 9.5 (PGP9.5) immunoreactivity, suggesting that LCN15 is produced in non-degenerated areas of the olfactory neuroepithelium. ELISA demonstrated that the concentration of LCN15 in the mucus was lower in participants with normal olfaction (≥ 50 years) and also tended to be lower in patients with idiopathic olfactory loss (≥ 50 years) than in participants with normal olfaction (< 50 years). Thus, LCN15 may serve as a biomarker for the activity of the Bowman’s glands.

We used an Acclaim PepMap® 100 (75 µm × 2 cm, Thermo Fischer Scientific) for the trap column and an ESI-column (75 μm × 12.5 cm, 3 μm, NTCC-360/75-3-125, Nikkyo Technos, Tokyo, Japan) for the analytical column. The chromatographic procedure comprised a 0.5 min hold at 2 % solvent B (0.1 % formic acid in acetonitrile, Thermo Fischer Scientific) and 78 min linear gradient from 2 % to 35 % solvent B. The next wash step was performed as 12 min linear gradient from 35 % to 75 % solvent B followed by a 9.5 min hold at 75 % solvent B. The solvent A consisted of 0.1 % formic acid (Thermo Fischer Scientific). Mass spectrometry analysis was carried out in a data dependent acquisition (DDA) mode with full scans (m/z 350-2,000) acquired at a mass resolution of 120,000. The spray voltage and ion transfer tube temperature were set to 1600 V and 275 °C, respectively. Among detected ions, charge states other than 2-4 were filtered out and the 10 most intense ions were selected for MS/MS analysis.
The tandem mass spectra were produced by collision induced dissociation (CID) method. An AGC target ion number for MS1 and MS2 were set to 4.0e5 and 1.0e4, respectively. The Cysteine carboxymethylation was specified as a fixed modification, and methionine oxidation as a variable modification. Scaffold (version 4.8.3, Proteome Software, Inc, Portland, OR) was used to integrate the proteomics data to compare the total spectrum counts. The thresholds for the protein false discovery rate (FDR), peptide FDR, and number of peptides per protein were set to 1.0 %, 0.1 %, and 2, respectively.

Preparation of hLCN15 protein
The primary amino acid sequence of mature hLCN15 (see below) was obtained from For the LC/MS/MS peptide mapping analysis, the same analytical conditions were used as described in 'Proteomics' above, and the recombinant human LCN15 sequence was used for sequence annotation.

Measurement of olfactory receptor activity
The luciferase reporter gene assay was performed as previously described

Bacterial growth inhibition tests
The tests were performed in collaboration with the OP Bio Factory Co., Ltd. (Okinawa, Japan).
To test the bacteriostatic activity of LCN15, the E. coli strain NBRC102203 was grown in 10 mL M9 minimal medium in culture tubes at 37 °C and 200 rpm for 18 h. The culture was then diluted to a concentration of 1×10 2 cells/mL with medium and transferred to 96-well dishes (Greiner, Wiesbaden, Germany) at 40 μL/well. The plates were incubated at 37 °C and 800 rpm.

Supplementary Figure Legends
The olfactory cleft mucosa of each sample was divided into four zones (dorsomedial, dorsolateral, ventromedial, and ventrolateral), and rectangular areas of each were analyzed (Fig.   4). We then compared the areas of PGP9.5-and LCN15-immunoreactivity in each rectangle.
Coefficients of determination between the two immunoreactive areas tended to be higher in the ventromedial (R 2 = 0.3985) and ventrolateral (R 2 = 0.2922) areas than in the dorsomedial (R 2 = 0.2314) and ventrolateral (R 2 = 0.1556) areas.
Correlation between the distribution of LCN15-negative/CK18-positive glands and PGP9.5immunoreactive area in the olfactory cleft mucosal zones. The olfactory cleft mucosa was divided into four zones, and the number of acini of LCN15-and CK18-positive glands were manually counted in each field. We calculated the ratio of the number of LCN15-negative glands/CK18-positive glands in each rectangle, then compared it with the area of PGP9.5 immunoreactivity. Coefficient of determinations between the parameters was less than 0.2 in each of four zones, indicating there was no statistical correlation.
Comparison of LCN15 concentration in OCLF of patients with normal olfaction and with idiopathic olfactory impairment. Each group was further divided into two subgroups by age  suggesting that LCN15 and albumin may affect odorant perception in these combinations.
However, all EC50 values in the LCN15 group were within the logEC50 95% CI of the albumin group, suggesting no statistical significance between LCN15 and albumin groups in any ORodorant conbination.
Bacteriostatic effects of LCN15 on the growth of E. coli. E. coli strain NBRC102203 was cultured in M9 minimal medium at a dilution of 1×10 2 cells/mL medium in 96-well dishes.